This invention relates to a new pigment in feed for salmonides, a new feed comprising this pigment and use of the pigment.
In feed for farmed salmon and trout pigment has to be added to obtain the desired colour of the fish flesh. The pigment mostly used is astaxanthin which corresponds to the pigment which is available in feed for wild salmonides. Also other pigments like for instance cantaxanthin, might be used. Such pigments are very unstable with regard to exposure to air and temperature as well as light. The pigments are therefore to a great extent degraded during feed processing and storage. These pigments are all carotenoids. This feed is mostly prepared from raw material not containing significant amounts of astaxanthin (i.e. white fish). Farmed salmon and trout is fed industrially manufactured feed where pigment is added.
Commercially available astaxanthin products are furthermore very expensive and their biological retention is very low. Astaxanthin is as mentioned above a rather unstable compound, which of course is a drawback. The low stability of astaxanthin is due to oxidation. Commercial pigment products are formulated in order to avoid or reduce oxidation. One typical formulation for astaxanthin is with gelatine and starch. The formulations used are often, however, not optimal with respect to biological availability of the pigment, and a new way of solving the stability problem, combining a high degree of stability with improved biological availability would be of great economical benefit to the aquaculture industry. A more stable pigment Is thus highly desired as this would give possibilities for making a formulation more optimal with regard to biological availability and consequently possibilities for considerably economic saving.
Thus it is a desire in the aquaculture industry to find more stable and biologically effective pigments useful in production of feed for salmonides.
The different salmonid species differ in their ability to utilise dietary carotenoid. Rainbow trout (Oncorhynchus mykiss) has been found to utilize the pigment in the feed more effectively than Atlantic salmon (Salmo salar) and sea trout (S. trutta).
Rainbow trout can also accumulate higher amounts of carotenoids in the flesh than Atlantic salmon and sea trout, but less than sockeye salmon (Oncorhynchus nerka) (Storebakken, T. and Ho, N. K., Aquaculture, vol. 100, (1992), p. 209).
In salmon, dietary astaxanthin and canthaxanthin are deposited more efficiently in flesh than in skin, which is in contrast to the rainbow trout (Schiedt, K. et al., Pure and Appl. Chem. 57 (1985) 685-692).
Synthetically produced astaxanthin is normally present in unesterified form (i.e. diol). This is also the form assumed that the pigment is converted to in the intestine before it is absorbed by the fish (O. J. Torrissen et al., Reviews in Aquatic Sciences, vol. 1, (1989) pp. 209-225). In nature astaxanthin is often present as diester.
Simpson, K. L. and Kamata, T., Proc. World Symp. on Finfish Nutr. and Fishfeed Technology, Hamburg. Jun. 20-23, 1978. Vol II. Berlin 1979, pp. 415-424, reported a study for pigmentation of rainbow trout comparing astaxanthin, astaxanthin ester and astacene. Astaxanthin, astaxanthin ester and astacene were extracted from shrimp coagulum. The pigments were dissolved in herring oil and added to the trout diet. When analysing the fish, no distinction was made between flesh and skin. The fish fed the diet consisting of astaxanthin ester contained much higher levels of total astaxanthin than others. This indicated that astaxanthin ester was more effective for the pigmentation of rainbow trout. However, on the same symposium Torrissen, O. and Braekkan, O. R. (Proc. World Symp. on Finfish Nutr. and Fishfeed Technology, Hamburg. Jun. 20-23, 1978. Vol II. Berlin 1979, pp 377-382) also demonstrated that astaxanthin was incorporated into the flesh of rainbow trout. These authors found that astaxanthin was more effectively incorporated in flesh than diesters and monoesters purified from the copepod, Calanus finmarchicus. 
According to O. J. Torrissen et al., Reviews in Aquatic Sciences, vol.1, (1989) pp. 209-225 (i.e.: Foss, P. et al., Aquaculture, vol. 65, (1987), p.293 and Storebakken, T. et al., Aquaculture, vol. 65, (1987), p. 279) synthetic astaxanthin diester (i.e. astaxanthin dipalmitate) seems to be absorbed less easy than free astaxanthin both in rainbow trout, sea trout and Atlantic salmon.
In crustaceans, a relatively large part of the astaxanthin is present in ester form. However, the pigment is more easily absorbed than what should be expected from the level of free astaxanthin. This is in O. J. Torrissen et al., Reviews in Aquatic Sciences, vol. 1, (1989) pp. 209-225 tentatively explained from other not identified compounds in crustacea which might contribute to enhanced absorption.
To summarise: as astaxanthin absorption in the intestine of the fish is assumed to involve free astaxanthin (i.e. diol) it is so far mainly considered that feeding with esters will give less biological absorption than feeding with free astaxanthin. This is supported by experiments with astaxanthin dipalmitate.
It is known that astaxanthin present as diester is more stable than free astaxanthin (Omara-Alwala, T. R. et al., J. Agric. Food Chem., vol. 33 (1985), p. 260 and Arai, S. et al., Aquaculture, vol. 66 (1987), p. 255.)
In the literature, dipalmitate is the predominant diester studied, and it is reported to give less pigmentation than the diol (Torrissen, O. and Braekkan, O. R.; Proc. World Symp. on Finfish Nutr. and Fishfeed Technology, Hamburg Jun. 20-23, 1978. Vol II. Berlin 1979, pp. 377-382, Storebakken, T. et al., Aquaculture, vol. 65 (1987), p. 279, Foss, P. et al., Aquaculture vol. 65 (1987), p. 293, Torrissen, O. J et al., CRC Crit. Rev. Aqua. Sci. vol. 1 (1989), p. 209. ). This is explained by a low degree of hydrolysis of the diester.
We have shown that by using a commercial lipase (Candida rugosa), after 42 hours the dipalmitate hydrolysed to 40% free astaxanthin. We synthesised several other diesters in order to study whether these hydrolysed faster than the dipalmitate. Example 1 shows that under the same experimental conditions the diester with elaidic acid (trans-C18:1) hydrolysed to a higher degree (73%) while a short-chain carboxylic acid (C10:0) hydrolysed somewhat slower, and a diester prepared with a concentrate of omega-3 fatty acids comprising approx. 50% EPA (all cis C20:5 n3) and approx. 35% DHA (all cis C22:6 n3) (in total more than 90% omega-3 fatty acids) (EPA+DHA) hydrolysed to free astaxanthin at less than half the rate of the dipalmitate.
To verify the hydrolysis data for these astaxanthin diesters obtained by using the commercial lipase, another similar experiment was performed by using enzyme isolated from the intestine of Atlantic salmon. This experiment very surprisingly gave opposite data than the data obtained in the experiment where the commercial lipase was used; i.e. the EPA+DHA diester was hydrolysed quickest and the dielaidate and the dipalmitate were hydrolysed slowest (see Example 2). Thus the inventors most surprisingly have found that if astaxanthin esterified with a concentrate of omega-3 polyunsaturated fatty acids is hydrolysed by enzyme from the intestine of salmon, a surprisingly fast hydrolysis to free astaxanthin is obtained compared to dipalmitate. Surprisingly, also diester with the short chain carboxylic acid (C10:0) was hydrolysed much faster than the dipalmitate, even though the rate of hydrolysis was significantly slower than hydrolysis of the EPA+DHA diester.
Based on these surprising data, the present inventors have found a astaxanthin EPA+DHA diester which most likely hydrolyses quickly to free astaxanthin when fed to the salmon and thus is effective for pigmentation of salmon. In Example 3 it is shown by feeding experiments that this statement is correct. In a similar way, the inventors have found that an astaxanthin diester with short chain carboxylic acids will be suitable as a pigment with good stability and a high potential for pigmentation of salmonides.
Furthermore, it was surprisingly found by the inventors that the EPA+DHA diester comprised acceptable stability properties for use in industrially manufactured feed without formulation with gelatine or starch (see Example 3). This was not expected, as omega-3 polyunsaturated fatty acids are unstable compounds.
In Example 3 pigmentation of salmon with the EPA+DHA diester was compared to pigmentation with a commercial free astaxanthin product (Carophyll Pink, Roche). Most surprisingly, it was found that the bioavailability as measured by astaxanthin uptake in salmon fillet was 41% higher in the fish fed the EPA+DHA diester compared with the fish fed the commercial pigment. Thus, feeding with this astaxanthin diester surprisingly gives enhanced biological absorption compared to free astaxanthin.
It is a main object of the invention to provide a pigment for feed to salmonides that is more stable and biologically effective than free astaxanthin and commercial pigments for salmonides.
Another object of this invention is to provide a pigment which can be added to the feed in less amounts than previously known pigments and still give a satisfactory pigmentation of the flesh.
This and other objects are achieved by the attached claims.